Tracking Dendritic Cells via Ultrasound-Guided Photoacoustic Imaging

Introduction: : Dendritic cell (DC)-based therapies offer a promising strategy for modulating immune responses in cancer, autoimmune disorders, and vaccine development. Despite encouraging preclinical results, clinical translation remains limited by the inability to monitor DCs post-delivery. A key determinant of therapeutic efficacy is the successful migration of DCs to draining lymph nodes, where DCs prime T cells and initiate adaptive immune responses. Current methods, such as histology and flow cytometry, are invasive, restricted to endpoint analysis, and fail to capture physiological changes within the lymph node. The lack of spatiotemporal information hinders evaluation of delivery efficiency, limits optimization of treatment strategies, and contributes to inconsistent therapeutic outcomes.
To address these limitations, we are developing a noninvasive imaging strategy based on ultrasound-guided photoacoustic (US/PA) imaging to track nanoparticle-labeled DCs and monitor lymph node physiology in vivo. US/PA imaging integrates the high spatial resolution of ultrasound with the molecular contrast of photoacoustics, enabling real-time visualization of DC migration. Beyond spatial tracking, this approach also captures physiological changes in the lymph node microenvironment during therapy—including alterations in oxygen saturation, blood flow, vascular remodeling, and lymph node size—parameters associated with immune activation and therapeutic engagement.
Overall, tracking labeled DC migration in conjunction with functional assessment of lymph node activity enables comprehensive evaluation of how delivery timing, spatial localization, and immune-associated lymph node changes influence therapeutic efficacy. These insights are essential for improving the design, effectiveness, and clinical translation of DC-based immunotherapies.

Materials and

Methods: : Hyperbranched gold nanoconstructs (HBGNCs), exhibiting broad near-infrared (NIR) absorption, were synthesized using a seed-mediated, surface-blocker-assisted growth method with 35 nm gold seeds and a silver halide complex to promote hyperbranched morphology [1]. HBGNCs were coated with polyethylene glycol. DCs were harvested and derived from the bone marrow of female C57BL/6 mice (6–8 weeks old; Jackson Laboratory). Bone marrow cells were cultured in RPMI medium supplemented with granulocyte-macrophage colony-stimulating factor (GM-CSF), initially at 20 ng/mL for 48 hours, increased to 40 ng/mL on day 4, and further raised to 60 ng/mL on day 6 [2]. On day 7, immature bone marrow DCs (BMDCs) were collected and incubated with HBGNCs at various optical densities (OD = 0, 1, 2, 4). After 24 hours, the labeled BMDCs were washed and collected. Viability of labeled BMDCs was assessed using the MTT assay. To induce maturation, the labeled BMDCs were treated with lipopolysaccharide for 48 hrs. For imaging studies, labeled BMDCs were injected into dome-shaped phantoms (40 µL, 8% gelatin) at varying cell densities and imaged using a Vevo LAZR system (20 MHz; 700–950 nm). For in vivo experiments, labeled BMDCs were delivered subcutaneously near the inguinal lymph node in C57BL/6 mice. Multi-wavelength, three-dimensional US/PA images were acquired using a motorized translation stage (step size: 0.152 mm). Both the injection site and the inguinal lymph node were imaged longitudinally at multiple time points, up to 6 days post-injection.

Results, Conclusions, and Discussions:: Results and

Discussion:
HBGNCs exhibited strong NIR absorbance between 700–900 nm, making them ideal for US/PA imaging. Transmission electron microscopy confirmed their uniform, branched morphology, with an average size of 87 nm ± 17.3 nm (Fig. 1A). Brightfield imaging of BMDCs labeled with varying concentrations of HBGNCs (0–4 OD) revealed increasing intracellular particle accumulation, with cells appearing progressively darker at higher labeling concentrations (Fig. 1B). The MTT assay demonstrated >95% viability across all concentrations, confirming the cytocompatibility of the HBGNCs (Fig. 1C). In in vitro phantom studies, US/PA imaging revealed a strong correlation between PA signal amplitude and the number of labeled cells per microliter, with signal intensity increasing proportionally with cell concentration (ranging from 62.5 to 500 cells/μL) (Figs. 1D, 1E). These data highlight the sensitivity and quantitative capability of the US/PA imaging platform in detecting labeled BMDCs. In in vivo studies, PA signal appeared in the draining lymph node by Day 2 post-injection, suggesting active trafficking and accumulation of DCs over time. No increased PA signal was observed in mice injected with unlabeled BMDCs, indicating that the observed signal in the experimental group was specifically from labeled BMDCs (Figs. 1F, 1G). Importantly, while lymph node enlargement was observed from Day 0 to Day 6 in both groups, no significant differences were observed in lymph node size between mice injected with labeled versus unlabeled BMDCs. This suggests that labeled BMDCs did not cause additional enlargement or structural changes (Figs. 1H).

Conclusion:
Our findings indicate that HBGNCs enable efficient and non-toxic labeling of DCs for US/PA imaging and tracking. Tracking DC migration, along with assessing lymph node activity, offers a promising non-invasive method for monitoring DC trafficking and physiological changes within the lymph node. This work contributes to the advancement of effective cancer treatment by guiding the development and evaluation of therapeutic strategies.

Acknowledgements and/or References (Optional):: [1] Kim. M, VanderLaan. D, Lee. J , Choe. A, Kubelick. K.P, Kim. J, and Emelianov. S.Y, Hyper-Branched Gold Nanoconstructs for Photoacoustic Imaging in the Near-Infrared Optical Window, Nano Letters 2023 23 (20), 9257-9265
[2] Sauter. M, Sauter. R.J, Nording. H, Olbrich. M, Emschermann.H , Langer. H. F, Protocol to isolate and analyze mouse bone marrow derived dendritic cells (BMDC), STAR Protocols, Volume 3, Issue 3, 2022